It has been a practice to use a Boyden chamber as an apparatus for detecting chemotaxis of cells in vitro. This apparatus has a structure partitioned into an upper chamber and a lower chamber by a filter having pores (diameter: 3 to 8 μm) through which cells can pass. A cell suspension is put into the upper chamber while a specimen solution containing a chemotactic factor is put into the lower chamber. Then cells migrating toward the chemotactic factor through the filter or cells appearing on the back face of the filter are counted. In this apparatus which is most commonly employed today, it is necessary to use ¼ to 1/20 ml of a cell suspension having a concentration of 1×106 cells/ml, i.e., corresponding to at least 5×104 cells. Although there scarcely arise any problems in case of analyzing cells which can be obtained in large quantities, it is highly laborious to obtain a necessary amount of cells occurring at a very limited level, for example, eosinophils contained in an amount of about 1 to 5% in peripheral leukocytes, basophils contained in an amount of 1% or less therein, or monocytes contained in an amount of about 1 to 2% therein. In case of using a small animal such as a mouse, blood can be collected in a highly limited amount, i.e., about 1.0 ml per animal at the largest. Moreover, cancer cells and some of cells existing in tissues can be hardly obtained in a large amount and it is therefore desired to examine the characteristics of these cells in microquantities. Furthermore, the Boyden chamber suffers from an additional problem that cells in the course of migration cannot be observed or counted thereby.
There have been marketed slide glass plates for qualitative analyses by which chemotaxis of cells can be observed at a level of several individuals. In such a slide glass plate, two grooves (wells) of 4 mm in width, 25 mm in length and 1 mm in depth are formed in both sides of a bridge (channel) of 1 mm in width on a glass slide (25×75 mm, 2 mm in thickness) for microscopes. Namely, two wells are connected to each other via the channel. A cell suspension is put into one well and a specimen solution containing a chemotactic factor is put into the other well. After covering with a glass plate, cells migrating from one well to the other well across the channel are observed. In this case, however, it is not assumed that the bridge forms a gap fitting for the diameter or derformability of the cells. Also, no groove through which the cells pass is formed in the channel. In addition, each well has a capacity of 100 μl. That is to say, it is needed to use at least 1/10 ml of a cell suspension per well. Also, there has been marketed another chemotaxis chamber having a similar structure in which two grooves (wells) are concentrically formed on a slide glass plate and a bridge (channel) is provided between these grooves (Dun Chemotaxis Chamber® manufactured by Waber Scientific). In this case, a cell suspension is put into the inner well while a specimen is put into the outer well. After covering with a glass plate, cells passing through the channel are microscopically observed. The channels are located lower by 20 μm than the cover glass and cells pass through the gap between them. The distance between the channel plane and the cover glass is set regardless of the diameter or deformability of cells and the channels have no grooves through which cells pass.
To measure blood rheology, Kikuchi et al. have proposed an apparatus having channels provided with a plural number of microgrooves formed on the surface of a single-crystal silicon substrate by using semiconductor fabrication techniques (Kikuchi, et. al., SPIE Vol.2978, 165–171 (1997); Kikuchi, et. al., Microvascular Research, Vol.44, 226–240 (1992); Kikuchi, et. al., Seibutsu Butsuri (Biophysics), Vol. 214, 254–258 (1997)). In this apparatus, it is intended to make a blood cell suspension flow due to a difference in pressure between both sides of the channel thereby observing and studying the blood flow. Although behaviors can be observed thereby at the cellular level, no structure for observing or measuring migration of blood cells by their own actions is employed in this idea.
Japanese Patent No. 2532707 has disclosed a blood circuit wherein large grooves each having an entrance port at one end and an exist port at the other end are formed in parallel and barriers partitioning these grooves are provided with microgrooves, by which the large grooves are connected to each other, orthogonally to the lines connecting the entrance ports to the exist ports. In this circuit, a blood sample is flown in one of the large grooves while a specimen containing a chemotactic factor is flown in the other groove. Then a portion of the blood sample is introduced into the microgrooves (channels) and cells passing through the microgrooves (channels) are detected to thereby examine the movements and functions of the cells or observe and measure the mobility thereof. Since flows in which the blood sample and the chemotactic factor-containing specimen are circulated are formed by the large grooves, this circuit has no well in which the blood sample or the chemotactic factor-containing specimen is contained in a resting state. In addition, the blood sample and the chemotactic factor-containing specimen are required each in a considerably large amount. Accordingly, this apparatus is unsuitable for studying movements of cells by their own actions with the use of microsamples.
There has been also known a blood filter wherein cells in blood are passed thorough microgrooves and thus the state of the blood cells during passage is observed (Japanese Patent No. 2685544). This filter consists of a first substrate made of a silicone substrate having microgrooves on the surface and a second substrate having a plane jointed to the surface of the first substrate. Blood cells pass through a space formed by the grooves of the first substrate at the interface of these substrates. To make the flow of blood cells in the microgrooves, it is needed to apply an external force by pressurizing, sucking, etc. Accordingly, the flow of the cells by their own actions cannot be observed by this apparatus. Namely, this apparatus has no well in which a blood sample or a specimen solution is contained in a resting state.
To fractionate cells depending on functional properties such as cell membrane hardness or cell deformability, there have been also known apparatuses by which cells to be fractionated are passed thorough channels having a large number of microgrooves to thereby divide the cells into passable ones and non-passable ones. For example, Japanese Patent No. 2685119 has proposed an apparatus wherein channels having different groove widths are formed in two stages for the multistage fractionation of cells. However, a solution containing cells is migrated under elevated pressure in this apparatus and thus migration of cells by their own actions cannot be understood thereby.
Moreover, there has been known a laminated microchannel array apparatus wherein substrates having channels provided with microgrooves are piled up each other so as to enable the filtration and fractionation of a large amount of a cell suspension (Japanese Patent Laid-Open No. 165062/1999). However, a solution containing cells is migrated under elevated pressure in this apparatus too and thus migration of cells by their own actions cannot be understood thereby.